Nitric oxide (NO) is associated with an array of diverse biological phenomena, such as inflammation, septic shock, adverse consequences of ischemia and reperfusion injury, hypotension, cell development, and apoptosis. Inflammation of a tissue in vivo is often characterized by the infiltration or presence of activated macrophages, which in turn produce NO as a mediator of vasodilation in the inflammation process. Thus, inflammation can be ameliorated by decreasing NO production by cells such as macrophages.
Chitosan is an acid-soluble polymer of xcex2-(1,4)-D-glucosamine. Chitin is a relatively insoluble, acetylated form of chitosan. Chitin, chitosan, and derivatives thereof are used in a number of industrial applications, including the production of viscosity control agents, adhesives, chromatography carriers, paper-strengthening agents, flocculent agents, food additives, drugs, and cosmetics.
The invention is based on the surprising result that chitosan and derivatives thereof can inhibit cellular NO production. This result was unexpected because it had been reported that chitosan stimulated NO production by rat macrophages in vitro (Peluso et al., Biomaterials 15:1215-1220, 1994). However, it appears that previous studies may have used preparations of chitosan that are contaminated with endotoxin or other agents that cause an increase in NO production. Therefore, the invention is directed to endotoxin-free preparations of chitin or chitosan.
Accordingly, the invention features a method of reducing NO production in a subject (e.g., a mouse, mammal, or human) by identifying a subject in which reduction of NO production is desired; and administering to the subject an amount of an endotoxin-free composition comprising chitosan sufficient to reduce NO production in the subject. The composition can be administered locally (e.g., at a site of inflammation in the subject, such as an arthritic joint), systemically (e.g., intravenously), or as part of an implantable device (e.g., a surgical prosthesis such as a wound dressing, or a device that provides for slow release of the composition). A reduction of NO production can be desirable when NO production is abnormally elevated; or when NO production is normal, but an NO production level below normal provides a benefit to the mammal.
The invention also includes a method of reducing NO production in a cell by contacting the cell with a composition comprising chitosan in an amount sufficient to reduce NO production in the cell.
The term xe2x80x9ccomposition comprising chitosanxe2x80x9d as used herein means (1) chitosan; (2) a derivative of chitosan, including partially acetylated chitosan, fully acetylated chitosan to form chitin, and chitosan formed of polysaccharides (e.g., of 5 to 10, or 5 or 6 sugar or glucosamine residues); and (3) a mixture of (1) or (2) or both, and containing one or more additives (e.g., a carrier) or other bioactive compounds, as discussed below. In general, chitosan is produced by deacetylating chitin. However, the above definition is meant to encompass both chitin and chitosan for efficiency""s sake.
The term xe2x80x9cendotoxin-freexe2x80x9d refers to a composition that does not test positive in any one of a number of endotoxin or lipopolysaccharide assays known in the art, such as described in Nakamura et al., Nephrol. Dial. Transplant. 15:1928-1934, 2000; Pang et al., J. Neurosci. Res. 62:510-20, 2000; or Schoeffel et al., Am. J. Surg. 180:65-72, 2000.
The methods of the invention can be used to treat or prevent any disease or condition in which NO production or its consequent physiological effects are undesirable. Such diseases or conditions are discussed below. In addition, the methods of the invention can be used for in vitro or in vivo animal model testing or screening of the efficacy of chitosan or derivatives thereof as a drug for treating various conditions, such as inflammation. Successful screening of effective and noneffective derivatives of chitin or chitosan is described in the Examples below (see, e.g., Tables 1 and 2).
Other features or advantages of the present invention will be apparent from the following detailed description, and also from the claims.
The invention relates to the administration of a composition including chitosan to a subject for the treatment or prevention of any condition or disease that is mediated by cellular NO production. Various methods for carrying out the invention are discussed below.
Specific diseases or disorders treatable with the composition include an inflammatory disease or disorder, hypotension, septic or traumatic shock, chronic hypotension, or priapism. Accordingly, the method of the invention can include administration of an amount of a vasoconstrictor, which together with chitosan or a derivative thereof, is effective to increase blood pressure. Suitable vasoconstrictors include epinephrine, norepinephrine, vasopressin, NG-monomethyl-L-arginine, NG-nitroarginine methyl ester, and prostagladin. The methods can also be used to inhibit smooth muscle cell relaxation in response to NO. Thus, in general, inhibition of NO production leads to many therapeutic benefits, either as the primary active ingredient in drug therapy or as an adjunct to another drug such as a steroid, antibody, or peptide hormone. In addition, the composition can be administered when NO production is normal, but an NO production level below normal provides a benefit to the mammal. For example, it may be desirable to decrease the normal vasodilation of endothelium in the kidney tubules because a patient is dehydrated.
Inflammation can involve a cell-mediated immune response, with release of toxic molecules such as NO. A cell-mediated immune response can be beneficial, e.g., for destroying infectious microorganisms such as bacteria and parasites, and for eliminating cancerous or infected cells. However, inflammation can become chronic, autoimmune, or detrimental, such as in asthma, cirrhosis, inflammatory bowel disease, and arthritis.
NO has been associated with the adverse effects of ischemic events. Ischemia or hypoxia is a particularly serious problem when it occurs in the heart, e.g., as a consequence of a myocardial infarct or after balloon angioplasty. Ischemia in the brain is also a serious but common problem associated with stroke. Thus, the methods of the invention can further include administration of a drug (e.g., tissue plasminogen activator or streptokinase) designed to release the blockage causing the ischemia.
NO is also an active neurotransmitter. Excessive production or activity of NO may result in neurological diseases, particularly those affecting the brain. Therefore, administration of the composition used in the method of the invention can treat neurological diseases. To facilitate crossing of the blood-brain barrier, if the composition is administered intravenously, the chitosan or derivatives thereof can be covalently linked to hydrophobic moieties or proteins that are known to cross the barrier. Alternatively, the composition can be administered intracranially or intraventricularly.
Chitin can be manufactured by the deproteination and decalcification of crab or shrimp shells (see, e.g., U.S. Pat. No. 3,903,268). Chitosan can then be obtained by deacetylating chitin with a hot alkali solution. Alternatively, chitin can be isolated from various fungal species, such as from a member of the genus Actinomucor. In addition, chitosan and derivatives thereof are available from known chemical vendors such as Sigma Chemical Co. (St. Louis, Mo.). Any preparation of chitosan should be tested to ensure that no detectable endotoxin is present in the preparation.
The composition of the present invention can be administered via any appropriate route, e.g. intravenously, intraarterially, topically, by injection, intraperitoneally, intrapleurally, orally, subcutaneously, intramuscularly, sublingually, intraepidermally, or rectally. It can be formulated as a solution, suspension, suppository, tablet, granules, powder, capsules, ointment, or cream. In the preparation of these compositions, a solvent (e.g., water or physiological saline), solubilizing agent (e.g., ethanol, Polysorbates, or Cremophor EL7), agent for making isotonicity, preservative, antioxidizing agent, excipient (e.g., lactose, starch, crystalline cellulose, mannitol, maltose, calcium hydrogen phosphate, light silicic acid anhydride, or calcium carbonate), binder (e.g., starch, polyvinylpyrrolidone, hydroxypropyl cellulose, ethyl cellulose, carboxy methyl cellulose, or gum arabic), lubricant (e.g., magnesium stearate, talc, or hardened oils), or stabilizer (e.g., lactose, mannitol, maltose, polysorbates, macrogols, or polyoxyethylene hardened castor oils) can be added. If necessary, glycerin, dimethylacetamide, 70% sodium lactate, a surfactant, or a basic substance such as sodium hydroxide, ethylenediamine, ethanolamine, sodium bicarbonate, arginine, meglumine, or trisaminomethane can be added. Pharmaceutical preparations such as solutions, tablets, granules or capsules can be formed with these components.
Chitosan or its derivatives can also be delivered in a composition that includes another drug. See, e.g., Knapczyk et al., xe2x80x9cPharmaceutical Dosage Forms with Chitosan,xe2x80x9d In: Chitin and Chitosan, Skjak-Braek et al., eds., Elsevier Applied Science, 1998, pp 665-669. This study and others, as well as the example below, indicate or suggest that chitosan and its derivatives are well tolerated and not cytotoxic in vivo. Thus, dosages of chitosan or derivatives thereof can be increased to effective levels within reason with little worry of toxicity.
The specific dose of the composition of the present invention is determined in consideration of the results of animal experiments and various conditions. More specific doses obviously vary depending on the administration method; the condition of the subject such as age, body weight, sex, sensitivity, food eaten, dosage intervals, medicines administered in combination; and the source, seriousness, and degree of NO-induced abnormality. The optimal dose and the administration frequency under a given condition must be determined by the appropriate dosage test of a medical specialist based on the aforementioned guide.
It is generally useful to test the efficacy of compositions, materials, or medical devices containing chitosan or derivatives thereof, before administering the composition into a human patient. For example, chitinous compositions can be tested for its ability to aid healing or treat a number of conditions in calves or cows as described in Minami et al., xe2x80x9cApplication of Chitin and Chitosan in Large Animal Practice,xe2x80x9d In: Advances in Chitin and Chitosan, Brine et al., eds., Elsevier Applied Science, New York, 1992, pp 61-69. Efficacy testing in small animals can be similarly accomplished as described in Okamoto et al., xe2x80x9cApplication of Chitin and Chitosan in Small Animals,xe2x80x9d In: Advances in Chitin and Chitosan, Brine et al., eds., Elsevier Applied Science, New York, 1992, pp 70-78; Johnson et al., xe2x80x9cIn vivo Tissue Response to Implanted Chitosan Glutamate,xe2x80x9d In: Advances in Chitin and Chitosan, Brine et al., eds., Elsevier Applied Science, pp 3-8, 1992; or Su et al., Biomaterials 18:1169-1174, 1997. For wound healing studies, the chitosan or its derivatives can be formed into surgical prosthetic sheets that are then used, for example, to close a hernia or replace a patch of skin. See Su et al., supra; Austin et al., Science 212:749-753, 1981; Su et al., Biomaterials 20:61-68, 1999; and Kifune, xe2x80x9cClinical Application of Chitin Aritificial Skin (Beschitin W), In: Advances in Chitin and Chitosan, Brine et al., eds., Elsevier Applied Science, pp 9-15, 1992. Alternatively, chitosan or its derivatives can be used to manufacture sutures that aid healing or decrease inflammation. See, e.g., Tachibana et al., Jap. J. Surg. 18:533-539, 1988; and Biagini et al., xe2x80x9cBiological Materials for Wound Healing,xe2x80x9d In: Advances in Chitin and Chitosan, Brine et al., eds., Elsevier Applied Science, pp 16-24, 1992. These testing procedures can be readily modified to specifically detect the amount of NO in tissue biopsies or an implanted device at the affected site by using known methods or the assays described in the example below.
Without further elaboration, it is believed that one skilled in the art can, based on the above disclosure and the description below, utilize the present invention to its fullest extent. The following examples are to be construed as merely illustrative of how one skilled in the art can practice the invention and are not limitative of the remainder of the disclosure in any way. Any publications cited in this disclosure are hereby incorporated by reference.